The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
A number of assays based on bioaffinity binding reactions or enzymatically catalyzed reactions have been developed to analyze biologically important compounds or their activity or their biological effect or its modulation from various biological samples (such as serum, blood, plasma, saliva, urine, faeces, seminal plasma, sweat, liquor, amniotic fluid, tissue homogenate, ascites, etc.), samples in environmental studies (waste water, soil samples), industrial processes (process solutions, products) and compound libraries (screening libraries which may comprise organic compounds, inorganic compounds, natural products, extracts of biological sources, biological proteins, peptides, or nucleotides, etc.). Some of these assays rely on specific bioaffinity recognition reactions, where generally natural biological binding components are used to form the specific binding assay (with biological binding components such as antibodies, natural hormone binding proteins, lectins, enzymes, receptors, DNA, RNA, LNA or PNA) or artificially produced binding compounds like genetically or chemically engineered antibodies, molded plastic imprint (molecular imprinting), other assays rely on activity or modulation of the activity of compounds present in sample or added into reaction (e.g. biologically active enzymes, chemical compounds with activity on biological molecules, enzyme substrates, enzyme activators, enzyme inhibitors, enzyme modulating compounds) and so on. Such assays generally rely on a label or a combination of multiple labels generating signals to quantitate the formed complexes after recognition and binding reaction. In heterogeneous assays a separation step (separations like precipitation and centrifugation, filtration, affinity collection to e.g. plastic surfaces such as coated assay tubes, slides or microparticles, solvent extraction, gel filtration, or other chromatographic systems, and so on) is generally required before e.g. the free or bound fraction of the label signal can be measured. In homogeneous assays the signal of the label or labels is modulated due to binding reaction or enzymatic activity or other measured effect and no separation step is needed before measurement of the label signal. Both in heterogeneous and homogeneous assays the measurement of the label signal from free or bound fraction of the label generally enables the calculation of the analyte or activity in the sample directly or indirectly, generally through use of a set of standards to which unknown samples are compared. Different binding assay methods have been reviewed recently in Principles and Practice of Immunoassay, 2nd ed., C. P. Price and D. J. Newman, eds., Palgrave Macmillan, Hampshire, UK, 2001; and The Immunoassay Handbook, 2nd ed. David Wild, ed., Nature Publishing Group, New York, N.Y., 2001.
High-affinity Binders to Small Molecules
Avidin (Green, N. M.; Adv. Protein Chem. 1975; 29: 85-133; and Wilcheck, M. and Bayer E. A. Methods in Enzymology: Avidin-Biotin Technology, 1990 Vol. 184) and streptavidin (Chaiet, I. and Wolf, F. J. The properties of streptavidin, a biotin-binding protein produced by Streptomycetes. Arch. Biochem. Biophys. 1964; 106: 1-5) have high binding affinity to biotin, the affinity being one of the strongest known reversible binding interactions. Both avidin and biotin and their derivatives are widely employed in biotechnology (Diamandis E P, Christopoulos T K. The biotin-(strept)avidin system: principles and applications in biotechnology. Clin Chem. 1991; 37: 625-636). The most commonly used high-affinity binders are monoclonal antibodies selected from hybridoma cultures and polyclonal antibodies. In addition to antibodies, there are numerous other examples of rapid and tight binders to small molecules, and in vitro evolution enables production of high affinity binders against almost any molecule (Lipovsek D, Pluckthun A. In-vitro protein evolution by ribosome display and mRNA display. J Immunol Methods. 2004; 290: 51-67; Pini A, Bracci L. Phage display of antibody fragments. Curr Protein Pept Sci. 2000; 1: 155-169; and Hoogenboom H R. Overview of antibody phage-display technology and its applications. Methods Mol Biol. 2002; 178: 1-37). Single-chain antibody mutants against small molecule fluorescent dye fluorescein (Boder E, Midelfort K, and Wittrup K; Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity, Proc Natl Acad Sci USA 2000; 97: 10701-10705) have been evolved in vitro with antigen-binding equilibrium dissociation constant Kd=48 fM and slower dissociation kinetics (half-time >5 days) than those for the streptavidin-biotin complex.
Fluorescence Resonance Energy Transfer
Fluorescence resonance energy transfer (FRET) (Förster, T. Intermolecular energy migration and fluorescence. Ann. Physik 1948; 2, 55-75.) (or Förster resonance energy transfer) describes an energy transfer mechanism between two fluorescent molecules or between a fluorescent and a non-luminescent molecule. A fluorescent donor is excited at its specific fluorescence excitation wavelength. By a long-range dipole-dipole coupling mechanism, this excited state is then nonradiatively transferred to a second molecule, the acceptor, which is luminescent and can emit at its specific emission wavelength, or the quencher, which is non-luminescent or luminescent. The donor returns to the electronic ground state. The mechanism is widely employed in biomedical research (reviewed by Selvin P R The renaissance of fluorescence resonance energy transfer. Nat Struct Biol 2000; 7: 730-734; and Lakowicz, J. Principles of fluorescence spectroscopy, 2nd edition. Plenum Press, New York, 1999).
The FRET efficiency is determined by the distance between the donor and the acceptor, the spectral overlap of the donor emission spectrum and the acceptor absorption spectrum, and the relative orientation of the donor emission dipole moment and the acceptor absorption dipole moment. The FRET efficiency E depends on the donor-to-acceptor distance r with an inverse 6th order law defined byE=1/(1+(r/R0)6)with R0 being the Förster distance of this pair of donor and acceptor at which the energy transfer efficiency is 50%. The Förster distance depends on the overlap integral of the donor emission spectrum with the acceptor absorption spectrum and their mutual molecular orientation.Self-quenched Fluorescent Oligomers and Oligomeric Substrates
Biooligomer derivatives, for example oligopeptide, oligonucleotide and oligosaccharide derivatives, containing both a fluorescent moiety and a quencher moiety covalently attached typically to different ends of the same oligomer molecule, are employed to measure hydrolysation or cleavage of the oligomer upon for example enzymatic or chemical activity. The hydrolysis and cleavage, resulting in increase in the distance between a fluorescent moiety and a quencher moiety, are accompanied by an increase in the fluorescence due to disruption of the intramolecular quenching of the fluorescent moiety. The spectral properties of the moieties do not necessarily need to be consistent with an energy transfer mechanism according to Förster requiring spectral overlapping between emission spectra of the fluorescent moiety (donor) and excitation spectra of the quencher moiety.
Self-quenched oligopeptide substrates, also called fluorogenic substrates, and their applications have been described e.g. by Lottenberg R, Christensen U, Jackson C M, Coleman P L Assay of coagulation proteases using peptide chromogenic and fluorogenic substrates. Methods Enzymol. 1981; 80: 341-61; and by Lew R A, Tochon-Danguy N, Hamilton C A, Stewart K M, Aguilar M I, Smith A I. Quenched fluorescent substrate-based peptidase assays. Methods Mol Biol. 2005; 298: 143-150. The use of specific quenched fluorescent oligopeptide substrates provides a rapid and sensitive method to measure peptidase activity, and is readily adaptable to high-throughput screening of potential peptidase inhibitors. A high throughput assay based on a peptide labelled with both a fluorescent europium chelate and a quencher has been described by Karvinen J, Hurskainen P, Gopalakrishnan S, Burns D, Warrior U, Hemmila I. Homogeneous time-resolved fluorescence quenching assay (LANCE) for caspase-3. J Biomol Screen. 2002; 7: 223-231. The principle of a peptidase assay based on quenched fluorescent substrate is illustrated in FIG. 1. In an intact fluorescent substrate the fluorescent label is quenched by the quencher, but when a peptidase cleaves the substrate the distance between the fluorescent label and the quencher increases recovering the fluorescence of the fluorescent compound. The measured signal is increased upon cleavage of the substrate.
Cleavage of the peptide by caspase-3 separates the quencher from the chelate and thus recovers fluorescence of europium chelate. A similar assay is possible by using a long-lifetime fluorescent metal-porphyrin label (O'Riordan T C, Hynes J, Yashunski D, Ponomarev G V, Papkovsky D B. Homogeneous assays for cellular proteases employing the platinum(II)-coproporphyrin label and time-resolved phosphorescence. Anal Biochem 2005; 342: 111-119). Phosphorescent platinum(II) coproporphyrin label was evaluated for the detection of cellular proteases by time-resolved fluorescence in homogeneous format. An octameric peptide containing the recognition motif for the caspase-3 enzyme was dual labelled with a new maleimide derivative of phosphorescent platinum(II) coproporphyrin label and with the non-luminescent quencher dabcyl. Donor-acceptor energy transfer and fluorescence quenching based assays have been described also for other enzymes: a protease related to apoptosis, helicase involved in DNA unwinding, and phosphatase having an important role in cellular signaling cascades (Karvinen J, Laitala V, Makinen M L, Mulari O, Tamminen J, Hermonen J, Hurskainen P, Hemmila I. Fluorescence quenching-based assays for hydrolyzing enzymes. Application of time-resolved fluorometry in assays for caspase, helicase, and phosphatase. Anal Chem 2004; 76: 1429-1436).
A cleavage assay can also be constructed using e.g. a terbium-chelate donor labelled streptavidin and using a biotinylated peptide substrate containing dabcyl as non-luminescent quencher or fluorescein as a luminescent acceptor at the other end of the peptide sequence. A similar cleavage assay using europium-chelate and donor labelled biotinylated peptide and streptavidin conjugate of XL665 luminescent acceptor is described in Kennedy M E, Wang W, Song L, Lee J, Zhang L, Wong G, Wang L, Parker E. Measuring human beta-secretase (BACE1) activity using homogeneous time-resolved fluorescence. Anal Biochem. 2003; 319: 49-55.
The principle of an assay with non-luminescent quencher is illustrated in FIG. 2, where the intact peptide contains both biotin and quencher moieties and is capable to bind to a fluorescent conjugate of streptavidin and quenches the fluorescence of the fluorescent label. When the peptide is cleaved the biotin and quencher moieties are separated and the quencher label is unable to bind to streptavidin and the fluorescence of the fluorescent label is not affected. Thus, the measured signal is increased upon cleavage of the substrate, because the cleavage prevents the quenching of the fluorescent label. The concentration of the fluorescent conjugate of streptavidin must be carefully adjusted because an excess of it results in a significant increase in the background signal.
FIG. 3 illustrates an assay based on a luminescent acceptor, where the substrate contains both biotin and acceptor moieties and is capable to bind to a donor conjugate of streptavidin. The sensitized acceptor emission is dependent on the proximity of donor and acceptor and only the acceptor present in an intact substrate is able to bind to streptavidin. Upon cleavage of the substrate the measured signal is decreased. The donor conjugate of streptavidin can be used in excess because signal without significant increase in the background signal. This method is used by Invitrogen (Carlsbad, Calif.; www.invitrogen.com) in their Lanthascreen concept based on terbium-chelate labelled streptavidin and biotinylated substrate labelled with fluorescein (http://www.invitrogen.com/-downloads/F-13279_LanthaScreen_Poster.pdf). The time-resolved FRET value is determined as a ratio of the FRET-specific signal measured with a 520 nm filter to that of the signal measured with a 495 nm filter, which is specific to terbium-chelate.
Fluorescence quenching assay based on an electrochemiluminescent label and luminescence quenching based on energy transfer is described in Spehar A M, Koster S, Kulmala S, Verpoorte E, de Rooij N, Koudelka-Hep M. The quenching of electrochemiluminescence upon oligonucleotide hybridization. Luminescence 2004; 19: 287-95. Interaction between electrochemically excited Ru(bpy)32+ and Cy5 in a hybridization assay on a chip was studied. The 3′ end of an oligonucleotide was labelled with Ru(bpy)32+ and the 5′ end of a complementary strand with Cy5. Upon the hybridization, the electrochemiluminescence (ECL) of Ru(bpy)32+ was efficiently quenched by Cy5 with a sensitivity down to 30 nmol/l of the Cy5-labelled complementary strand. The quenching efficiency is calculated to be 78%.
Quantitative 5′-nuclease based polymerase chain reaction assay (TaqMan; Applied Biosystems, Foster City, Calif.) is a nucleic acid sequence detection method wherein a single-stranded self-quenching oligonucleotide probe, containing both a fluorescent moiety and a quencher moiety, is cleaved by the nuclease action of nucleic acid polymerase upon hybridisation during nucleic acid amplification (Lie Y S, Petropoulos C J. Advances in quantitative PCR technology: 5′ nuclease assays. Curr Opin Biotechnol. 1998; 9: 43-48; and Orlando C, Pinzani P, Pazzagli M. Developments in quantitative PCR. Clin Chem Lab Med. 1998; 36: 255-269).
Molecular beacons are single-stranded oligonucleotide hybridization probes that form a stem-and-loop structure (Tan W, Wang K, Drake T J. Molecular beacons. Curr Opin Chem Biol. 2004; 8: 547-553; and Tan W, Fang X, Li J, Liu X. Molecular beacons: a novel DNA probe for nucleic acid and protein studies. Chemistry 2000; 6: 1107-1111). The loop contains a nucleic acid probe sequence that is complementary to a target sequence, and the stem is formed by the annealing of complementary arm sequences that are located on either side of the probe sequence. A fluorescent moiety is covalently linked to the end of one arm and a quencher is covalently linked to the end of the other arm. Due to the proximity of a fluorescent moiety and a quencher moiety molecular beacons do not fluoresce when they are free in solution. However, when they hybridize to a complementary nucleic acid strand containing a target sequence they undergo a conformational change increasing the distance between fluorescent moiety and the quencher moiety that enables the probe to fluoresce. In the absence of a complementary target sequence, the beacon probe remains closed and there is no fluorescence due to intramolecular quenching.
Selective cleavage of internucleotide bonds of self-quenched single-stranded oligonucleotide probes, which contain one or more ribonucleotides, by RNase H upon double-stranded helix formation subsequent to hybridisation to target is another method of target sequence detection (Rizzo J, Gifford L K, Zhang X, Gewirtz A M, Lu P. Chimeric RNA-DNA molecular beacon assay for ribonuclease H activity. Mol Cell Probes 2002; 16: 277-283). Yet another method is to use a self-quenched single-stranded oligonucleotide cycling probe, which is cleaved by a double-stranded selective exonuclease upon hybridisation to target sequence. Examples of nuclease-based assays are found in e.g. Till B J, Burtner C, Comai L, Henikoff S. Nucleic Acids Res. 2004; 32: 2632-2641.
Self-quenched fluorescent probes are also used to monitor nucleic acid amplification process in a thermal cycler; for example in quantitative polymerase chain reaction the amount of fluorescence at any given cycle, or following cycling, depends on the amount of specific product. The self-quenched single-stranded fluorescent probes, for example molecular beacons or Taqman probes, bind to the amplified target following each cycle of amplification and the resulting signal upon hybridisation, and in case of Taqman probes upon cleavage, is proportional to the amount of the amplified oligonucleotide sequence. Fluorescence is measured during each annealing step when the molecular beacon is bound to its complementary target or after elongation step when the Taqman probe is cleaved. The information is then used during quantitative PCR or RT-PCR (reverse transcriptase PCR) experiments to quantify initial copy number of amplified target nucleic acid sequence. For endpoint analysis, PCR or RT-PCR reactions containing molecular beacons can be run on any 96-well thermal cycler and then read in a fluorescence reader.
Fluorescent oligosaccharide substrates and their use in fluorescence quenching assay has been described in Cottaz S, Brasme B and Driguez H, A fluorescence-quenched chitopentaose for the study of endo-chitinases and chitobiosidases. Eur. J. Biochem. 2000; 267: 5593-5600.
Non-fluorescent acceptor labels and their use in fluorescence quenching assays with short-lifetime fluorescent dyes have been described e.g. in U.S. Pat. No. 6,828,116.
Ribonuclease detection using dual-labelled quenched fluorescent oligonucleotide containing both short-lifetime fluorescent dye and non-luminescent acceptor has been described in US 2004/0137479.
Fluorescent quenching assay for protein kinase based on fluorescent labelled substrate and phosphate specific binder labelled with non-luminescent acceptor is described in US 2004/024946.
Fluorescence quenching assays based on both fluorescent streptavidin-coated microspheres and conjugates of small-molecule fluorescent dyes in combination with both non-luminescent acceptor dye and quencher polymer have been described in US 2003/0054413.
Fluorescence quenching assay based fluorescent streptavidin-coated microsphere and biotinylated non-luminescent acceptor labelled protease substrate for measurement of protease activity has been described in US 2005/0014160.
Protease activity assay based on dual-labelled fluorescent protein substrate containing binding moiety for purification and separation is described in US 2005/0214890.
In all of the aforementioned examples a fluorescent moiety or a fluorescent compound (donor) is used in combination with either non-luminescent compound (quencher) or luminescent compound (acceptor), respectively, and the donor compound is capable of transferring energy either to a quencher or to an acceptor, respectively, said energy transfer being dependent on the distance between the donor and quencher or acceptor.
In all cases the donor is excited directly by light or electrochemically and, in case of a non-luminescent acceptor, it's the donor's own light emission (fluorescence) is measured or in case of luminescent acceptor, the sensitized emission of an acceptor (originating from energy transfer) is measured.
Homogeneous Bioassay Technologies
Homogeneous assay methods (Ullman E F, J Chem Ed 1999; 76: 781-788; Ullman, E F, J Clin Ligand Assay 1999; 22: 221-227) based on photoluminescence have received much attention, since several types of physical and chemical interactions can be employed to modulate the emission of photoluminescent labels due to formation of specific immunological complexes. The commonly employed methods are based on polarization of the emitted light or nonradiative energy-transfer between two photoluminescent compounds or between a photoluminescent and a non-luminescent compound (Hemmilä I, Clin Chem 1985; 31: 359-370). Fluorescence properties of two fluorescent compounds were employed in a homogeneous immunoassay in late 1970's when Ullman et al. demonstrated, that fluorescence energy transfer between a fluorescein donor and tetramethylrhodamine acceptor pair could be employed to construct both competitive and non-competitive immunoassays (Ullman E F et al. J Biol Chem 1976; 251: 4172-4178; Ullman E F & Khanna P L, Methods Enzymol 1981; 74: 28-60). The energy transfer was measured from decrease in the fluorescence of the donor, which limited further improvements in sensitivity. Increase in the fluorescence of the acceptor was not practicable, since only a little increase in a sensitized acceptor emission could be observed over autofluorescence, light scattering or absorbance of biological sample matrices and the direct emission of the donor at acceptor-specific wavelength.
Many compounds and proteins present in biological fluids or serum are intrinsically fluorescent, and the use of conventional fluorophores leads to serious limitations of sensitivity (Wu P and Brand L, Anal Biochem 1994; 218:1-13). Another major problem with homogeneous fluorescence techniques is the inner filter effect and the variability of the optical properties of a sample. Sample dilution has been used to correct this drawback, but always at the expense of analytical sensitivity. Feasibility of fluorescence energy transfer in immunoassays was significantly improved when fluorescent lanthanide cryptates and chelates with long-lifetime emission and large Stokes' shift were employed as donors in the 1990's (Mathis G, Clin Chem 1993; 39: 1953-1959; Selvin P R et al., Proc Natl Acad Sci USA 1994; 91: 10024-10028; Stenroos K et al., Cytokine 1998; 10:495-499; WO 98/15830; U.S. Pat. No. 5,998,146; WO 87/07955). Feasibility of the label technology in dissociation reactions, e.g. cleavage assays has also been described (Karvinen J et al., J Biomol Screen 2002; 7: 223-231).
Time-resolved fluorescence detection of sensitized emission allowed elimination of autofluorescence (Soini E and Kojola H Time-resolved fluorometer for lanthanide chelates—a new generation of nonisotopic immunoassays. Clin Chem 1983; 29: 65-68). Dual signal ratio measurement (U.S. Pat. No. 5,527,684; Mathis, G, Clin Chem 1993; 39: 1953-1959) corrected the variability of optical properties of the sample in homogeneous assay. Fluorescence of the compounds and proteins present in biological fluids has a short lifetime and the use of long-lifetime labels combined with time-resolved detection of the sensitized (prolonged lifetime) acceptor emission allowed minimization of the assay background and improved signal to background ratio. The variability of absorption of excitation light at 337 nm was corrected by measuring the emission of the donor at 620 nm and using the ratio of the energy transfer signal at 665 nm and the emission at 620 nm to generate a quantity that is independent of the optical properties of the serum sample. Homogeneous time-resolved FRET based bioaffinity assays using long-lifetime fluorescent nanoparticles have been described in WO 02/044725 and by Kokko L, Sandberg K, Lövgren T and Soukka T, Europium(III) chelate-dyed nanoparticles as donors in a homogeneous proximity-based immunoassay for estradiol Anal Chim Acta 2004; 503: 155-162. In the latter publication it is described that multiple lanthanide chelates inside a single particulate can participate simultaneously in energy transfer to a single acceptor. However, still only a small part of the lanthanide chelates inside the entire particulate can participate in an energy transfer to a single acceptor and thus the entire fluorescence of a particulate label cannot be quenched by a single acceptor moiety. The same problem is also encountered when lanthanide chelates are incorporated in a solid phase.
Separation-free assay technologies based on confocal detection of photoluminescent labels bound on particulate carriers have been introduced as an alternative to real homogeneous assays (Saunders G C et al., Clin Chem 1985; 31:2020-2023; Frengen J et al., Clin Chem 1993; 39:2174-2181; Fulton R J et al., Clin Chem 1997; 43:1749-1756). In recent years, the technology has been developed, and some novel carrier-based immunoassays can be considered as homogeneous assays, since they are practically similar to perform (Hänninen P et al., Nat Biotechnol 2000; 18:548; U.S. Pat. No. 5,891,738; Schaertl S et al., J Biomol Screen 2000; 5:227-238), although the actual signal of the label is not modulated, but the unbound labelled component is spatially excluded from measurement. These assays are otherwise comparable to homogeneous assays, but measurement is relatively slow, since carrier particles have to be either actively scanned or passively diffuse to a focal point, and a signal associated to several carrier particles is required for reliable measurement (Waris M E et al., Anal Biochem 2002; 309: 67-74). To avoid sterical hindrance in binding at least one of the labels, preferably both labels of a label-pair should be of small molecular size.
In most of the conventional homogeneous fluorescence assay technologies, the performance has still severe limitations: the sensitivity is limited by interferences from matrix components and optical properties of matrices, e.g. urine, saliva, serum, plasma or whole blood, to fluorescence yield and level of background, and by the attainable degree of fluorescence modulation, e.g. quenching, enhancement, energy transfer or polarization (Hemmilä I, Clin Chem 1985; 31: 359-370). In practice, only wavelengths in the range 600 to 1100 nm, or more preferably in the near infrared, in a wavelength range 650 to 950 nm, are practicable when a whole blood sample is employed (Chance B, Photon Migration in Tissues, pp. 206; Kluwer Academic/Plenum Publishers, 1990, New York).
Homogeneous luminescence-based whole-blood assay based on FRET and up-conversion photoluminescence is described in WO 2004/086049. Both the excitation and the measurement of sensitized acceptor emission have to be performed at far-red and infrared wavelengths where the sample is transparent, in this case at wavelengths 900-1000 nm and approximately 580-640 or 690-750 nm.